Throughout this application various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of each section and in the body of the text.
The interaction of antibody-antigen complex with cells of the immune system results in a wide array of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulating lymphocyte proliferation and antibody secretion. All these interactions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. It is now well established that the diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of Fc receptors. Considerable progress has been made in the last several years in defining this heterogeneity for IgG and IgE Fc receptors (Fc.gamma.R, Fc.epsilon.R) through their molecular cloning. Those studies make it apparent that Fc receptors share structurally related ligand binding domains, but differ in their transmembrane and intracellular domains which presumably mediate intracellular signalling. Thus, specific Fc.gamma.Rs and Fc.epsilon.R has also revealed at least one common subunit among some of these receptors.
It was recently observed that a family of disulfide-linked dimers are shared by Fc receptors and the T cell antigen receptor (TCR). Comparison of the genes for Fc.epsilon.RI(Fc.gamma.RIII).gamma. and TCR.zeta. chain indicates that they belong to the same family and have been generated by duplication. Both genes are located on mouse and human chromosome 1 and show an analogous organization of their exons. In both genes, the leader peptide is encoded by two exons, the second of which also contains the short extracellular domain, the hydrophobic transmembrane region, and the beginning of the cytoplasmic tail. The following exons, exons 3-5 and exons for .gamma. and .zeta., respectively, encode the remainder of the cytoplasmic tail. Furthermore, a high level of homology between the two genes is found in three of their respective exons, at the DNA and protein level (both about 50%). Finally, both .gamma. and .zeta. polypeptides use homologous cysteines essential for the surface expression of their respective receptors.
The detection of transcripts for .zeta. chains in TCR-, CD3-NK cells led to the finding that human Fc.gamma.RIIIA.alpha. from NK cells physically associates with .zeta.-.zeta. homodimer and with .zeta.-.gamma. heterodimer. So far, three different dimers have been identified in Fc receptor complexes: .gamma.-.gamma..zeta.-.zeta. and .zeta.-.gamma.. These dimers are also part of the TCR complex and probably mediate similar functions. There is a third member of the same family, TCR.eta. which is generated by alternate splicing from the same gene as TCR.zeta.. The dimers .eta.-.eta., .eta.-.zeta., and .eta.-.gamma. apparently are only associated with TCR, and so far there is no evidence that they associate with Fc receptor structures. Possibly, new members of the same family will be identified that form part of Fc receptor complexes.
Fc receptors (FcRs) for IgG and IgE couple humoral and cellular immunity by directing the interaction of antibodies with effector cells. These receptors are present on most effector cells of the immune system and mediate phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), activation of inflammatory cells and many of the biological sequelae associated with antibody-dependent immunity (reviewed in Ravetch and Kinet, 1991; Beaven and Metzger, 1993). Intensive analysis of the genes and proteins encoded by this family of receptors has revealed a structural heterogeneity for these receptors which mirrors the functional diversity mediated by these cell surface molecules (Ravetch, et al., 1986).
The high affinity FcR for IgE, Fc.epsilon.RI, is found on mast cells and basophils, and is responsible for the degranulation of these cells in response to crosslinking by antigen (reviewed in Parker, 1987). It is the receptor primarily responsible for triggering both peripheral and systemic anaphylaxis. In addition to the well known pathological response of these cells when activated by allergen, Fc.epsilon.RI has been associated with host resistance to parasitic infections (Matsuda et al., 1990). In contrast to the restricted expression of Fc.epsilon.RI, FcRs for IgG, Fc.gamma.Rs, are found on most cells of the hematopoietic lineage, and mediate both high and low affinity binding to IgG. The high affinity receptor, Fc.gamma.RI, binds monomeric IgG and is expressed exclusively on macrophages and neutrophils. It is capable of mediating ADCC and phagocytosis in response to crosslinking by antibody (Askenase and Heyden, 1974, Heusser, et al. 1977; Diamond et al. 1978). The low affinity receptors for IgG, Fc.gamma.RII and Fc.gamma.RIII, are responsible for effector cell responses to immune complexes and represent the Fc.gamma.Rs primarily involved in the inflammatory response in vivo. Fc.gamma.RII is widely expressed on haematopoietic cells and functions as an inhibitory receptor on B cells (Uhen, et al., 1985; Kurosaki, et al. 1993), while on cells of the myeloid lineage and on platelets, Fc.gamma.RII triggers ADCC, phagocytosis and the release of inflammatory mediators when crosslinked by immune complexes (Nathan, et al. 1980). These disparate functions result from the genetic heterogeneity of Fc.gamma.RII, as well as alternative splicing of its mRNA to generate proteins with distinct intracellular domains (Stuart, et al., 1989; Brooks, et al., 1989; Qiu, et al.,1990). Fc.gamma.RIII is restricted in its expression to NK, macrophage, neutrophils and mast cells, and mediates effector responses when crosslinked by immune complexes (Weinshank, et al., 1988; Perussia, et al., 1989). It is the sole FcR on NK cells, mediating all the antibody-dependent responses on those cells. In addition to these well-characterized effector cell pathways, Fc.gamma.RIII has been found on immature (day 15) thymocytes, where it has been postulated to function in early thymocyte development (Rodewald, et al., 1992).
Molecular characterization of the genes and protein products for Fc.gamma.RIII and Fc.epsilon.RI revealed that these two receptors were homologous (Ravetch and Anderson, 1989; Ravetch and Kinet, 1991) and required the identical subunit, the .gamma. chain, for efficient cell surface expression (Ra, et al. 1989; Kurosaki and Ravetch, 1989). This homodimeric protein not only mediates assembly of these receptors by preventing the degradation of the ligand binding .alpha. subunit in the endoplasmic reticulum (Weissman, et al., 1989; Kurosaki, et al. 1991), it is also critical for transducing signals into the cell interior resulting in cellular activation through a tyrosine kinase-dependent pathway (Romeo and Seed, 1991; Wirthmueller, et al., 1992). In murine macrophages, neutrophils, mast cells and basophils, the .gamma. chain is necessary for surface expression of Fc.gamma.RIII and Fc.epsilon.RI. In NK cells, a homologous chain, the .zeta. chain, first described as a component of the TCR/CD3 complex, is also expressed and forms heterodimers with the .gamma. chain (Kurosaki and Ravetch, 1989; Lanier, et al., 1989). Based on reconstitution studies and in vitro experiments, the murine .zeta. chain alone cannot substitute for .gamma. chain, due to a single amino acid substitution in the transmembrane domain of this .zeta. replacing a leucine for an isoleucine (Kurosaki and Ravetch, 1989; Kurosaki et al., 1991). This change in the transmembrane domain greatly diminishes the association of .zeta. chain with the ligand binding a chain. The .gamma. chain has also been found to be associated with the TCR/CD3 complex (Mercap et al., 1990), although its specific function, distinguishing it from the homologous .zeta., chain has yet to be determined. Although not required for its surface expression or ligand binding in transfected fibroblasts, the .gamma. chain has been found to be associated with FC.gamma.RI in the human monocytoid line U937 (Ernst, et al. 1992), where it may function as a signal transducing subunit.
The tissue deposition of immune complexes in diseases as diverse as rheumatoid arthritis, systemic lupus erythematosis, glomerulonephritis, and vasculitis is widely recognized as a major pathogenic factor triggering the inflammatory cascade, leading to tissue damage and its subsequent morbidity and mortality. The most widely employed experimental model for the study of the pathological effects of antibody-antigen interaction is the Arthus phenomenon, first described by Maurice Arthus in 1903 (1). It was first characterized as the acute local inflammation and hemorrhage produced when an intradermal injection of horse serum was administered to previously sensitized rabbits, and its manifestations are a direct result of immune complex formation and deposition: edema due to increased vascular permeability and local mediator release, neutrophil infiltration in response to the local formation of chemotactic peptides, hemorrhage due to damage to the blood vessel wall, and in severe cases, tissue damage produced by the release of lysosomal enzymes. The study of the mechanisms and inhibitors of this reaction thus has broad relevance to the understanding of immune complex-mediated diseases and has provided important insights into the understanding of the process of inflammation.
Because the induction of the direct Arthus reaction suffers from substantial intra- and interspecies variability in the immunologic responsiveness to a foreign antigen, a number of experimental variants of the original Arthus reaction have been developed. The one which best minimizes the difficulties with reproducibility and generalizability is the reverse passive Arthus reaction, in which heterologous antibody is injected into the skin and cognate antigen is injected intravenously; immune complexes are formed locally in the skin as circulating antigen diffuses into the tissue and binds to its antibody. Since it is independent of host response to specific antigens in the levels and specificity of antibodies generated, this variant maximizes the detection of host factors necessary to the inflammatory response and as such has allowed elucidation of the many elements contributing to this complex cascade.
The first definitive experiments demonstrating that serum antibody was necessary to the Arthus reaction were performed by Opie in 1924 (2); subsequent experiments by Culbertson, Cannon and Marshall, Fishel and Kabat, and Benacerraf and Kabat (3) correlated the actual quantity of antibody with the intensity of the reaction. Using fluorescent antibody techniques, Cochrane and Weigel in 1958 (4) demonstrated the presence of both antigen and antibody in histologic lesions, supporting the hypothesis first put forth by Opie (2) more than a quarter of a century earlier that the Arthus reaction was produced by the local formation of antigen-antibody complexes.
That polymorphonuclear leukocytes play a critical role in the Arthus reaction was demonstrated independently in the 1950's by Stetson, Humphrey and Cochrane, et. al. (5). Animals which were depleted of neutrophils with either nitrogen mustard or anti-neutrophil antiserum showed markedly reduced Arthus reactions, despite the continued presence of antibody-antigen complexes. In 1964, Ward and Cochrane (6) demonstrated the integral role of complement to the production of the Arthus reaction by pre-treating animals with cobra venom factor, which cleaves the C3 component of complement and inactivates the cascade. In the absence of complement, neutrophil infiltration was substantially attenuated, consistent with the key role of complement in inflammatory activation. Once it became known that immune complexes can bind and activate complement directly via the "classical pathway" (7) and that activated complement components are themselves potently chemotactic for neutrophils (8), a model of immune complex-triggered inflammatory disease was proposed which has persisted to this day.
In this model, antibodies bind to their antigen to form immune complexes, which results in complement binding and activation via the "classical pathway". The resulting chemotactic peptides cause neutrophil invasion and activation, with subsequent discharge of granules (degranulation) and release of inflammatory mediators. The direct consequences of this cascade are the classical symptoms of inflammation--edema, hemorrhage and tissue destruction. However, this model has not addressed the potential role of specific cell-surface receptors known to bind antibody-antigen complexes and activate effector cells. These well-defined receptors, collectively known as Fc receptors for their binding of the Fc portion of antibodies, mediate macrophage, neutrophil, NK cell and mast cell activation in vitro and are capable of triggering many of the responses classically associated with inflammation (9).
Fc receptors are members of the immunoglobulin superfamily and exist as membrane-associated glycoproteins. Distinct receptors are expressed for each isotype of antibody. Among the IgG Fc receptors, three classes of molecules have been defined, varying in structure and affinity for IgG. Fc.gamma.RI, present on monocytes and macrophages, is the only Fc receptor capable of binding monomeric antibody, due to its relatively high ligand affinity. Fc.gamma.RII and Fc.gamma.RIII are both low affinity receptors and will only bind antibody in the form of immune complexes. Fc.gamma.RII is expressed widely on hematopoietic cells, whereas FC.gamma.RIII expression is generally limited to monocytes, NK cells, neutrophils, and mast cells.
Fc receptors are generally hetero-oligomeric receptors, composed of a ligand binding subunit .alpha., and in the case of Fc.epsilon.RI, Fc.gamma.RI and Fc.gamma.III of a dimeric .zeta. or .gamma. chain, required for surface expression and signal transduction. While the .alpha. subunits of murine Fc.gamma.RII and III are nearly identical in their extracellular domains, they have distinct transmembrane and intracytoplasmic regions, which mediate their interaction with associated subunits and thus result in the activation of different signaling pathways (9). Dissecting the role of individual Fc receptors in vivo has been complicated by the overlapping expression of this large family of related receptors, each of which can bind immune complexes and mediate effector cell response.
An oligopeptide which blocks immune complex binding to immunoglobulin Fc receptors is disclosed in U.S. Pat. No. 4,686,282 (Hahn, 1987).
U.S. Pat. No. 5,198,342 (Maliszewski, 1933) discloses DNA encoding IgA Fc receptors.